BACKGROUND: Malignant transformation is characterized by enhanced uptake and utilization of glucose, which results in the persistent activation of aerobic glycolysis - a phenomenon known as the Warburg effect. Accumulating evidence suggests that the metabolic changes associated with tumor development may not simply be a consequence of increased nutrient demand, but act as causal contributors to disease development or progression. Consistent with this, altered metabolite profiles have been implicated in the epigenetic control of gene expression, including activation of oncogenes and repression of tumor suppressors, which points to an important role for metabolites as modulators of chromatin integrity in tumor cells. Notably, chromatin does not only determine gene expression and epigenetic integrity of nuclear DNA, but directly contributes to the repair of genomic lesions. The link between the tumor-associated increase in glucose metabolism, thus, presents a plausible means through which malignant transformation may alter DNA double-strand break (DSB) repair and genome stability. However, how metabolic changes promote malignant transformation remains unclear. OBJECTIVE AND RESULTS: A significant fraction of tumors is characterized by the deregulation of growth and survival genes rather than apparent defects in genome maintenance genes. Nevertheless, such tumors often carry deleterious genomic mutations and/or chromosomal aberrations. To determine if and how tumorigenesis may modulate genome maintenance, we will generate a reporter system to dissect the impact of malignant transformation on the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Our experimental approach is based on a previously described cell line, where cellular transformation can be induced in vitro via the aberrant activation of v-Src kinase, an oncogene that promotes growth factor receptor kinase signaling. Specifically, normal mammary epithelial cells (MCF10A) that contain a stably integrated v-Src gene fused to the ligand-binding domain of the estrogen receptor (ER-Src) undergo v-Src-dependent transformation within 24 to 36 hours after administration of the ER ligand 4-hydroxy-tamoxifen (4-OHT). To compare DSB repair efficiency before and after cellular transformation, MCF10A-ER-Src cells will be stably transfected with reporter constructs that allow for the detection of cells that have undergone NHEJ or HR in response to I-SceI endonuclease-mediated DSB induction. Both reporters rely on DSB repair-dependent expression of GFP, and repair efficiency can, thus, be measured as the fraction of GFP+ cells following I-SceI induction. Using this experimental system we will address the following specific aims: 1.) Determine the effect of malignant transformation on DSB repair outcome, and 2.) Characterize transformation-induced metabolic and epigenetic changes in the context of DSB repair. IMPLICATIONS: This project is expected to determine the involvement of metabolites in DSB repair, which has significant implications for the therapeutic modulation of repair outcome. Changing nutrient availability, cellular metabolism and/or metabolite abundance may eventually be utilized to either promote DNA repair and, thus, prevent damage accumulation, or to impair DSB repair, thus enhancing genotoxic therapy in cancer treatments.